Antibiotic Nystatin A1 is the main component of antibiotic complex Nystatin, a group of tetraeno-diene polyene macrolides produced by Streptomyces noursei (E. L. Hazen, R. Brown, Science 112, 112, 1950; E. L. Hazen, R. Brown, Proc. Soc. Exptl. Biol., 76, 93, 1951; A. H. Thomas and in., J. Chemother., 216, 367, 1981; A. H. Thomas et al., Analyst 107, 849, 1982) which in addition to Nystatin A1 (Borowski et al., Tetrahedron Lett. 8, 685, 1971) also contains Nystatin A2 (J. Pawlak et al., Polish J. Chem. 79, 1673, 2005) and Nystatin A3 (J. Zielinski et al., J. Antibiot. 41, 1289, 1988). Nystatin A1 is currently commercially available in acceptably pure form, manufactured by Bristol-Myers-Squibb in USA, as well as by other companies. Nystatin A1 and other components of antibiotic complex Nystatins are also produced by Streptomyces noursei var. Polyfungini named polyfungin (N. Porowska et al., Rec. Tray. Chem. 91, 780, 1971). This complex contains additionally a new component called polyfungin B (Zielinski et al., J. Antibiot. 32, 565, 1979). Among tetraeno-diene polyene macrolides, well identified compound is also Amphotericin A (P. Sowinski et al., J. Antibiot. 38, 175, 1985), produced together with Amphotericin B by Streptomyces nodosus. According to our invention, spatially hindered derivatives of Nystatin A1 and process of their preparation also apply to the other above-mentioned tetraeno-dienes.
Chemotherapy of fungal infections is one of the most difficult and not yet successfully solved problems in modern medicine. This is a consequence of the fact that both, pathogenic fungal organisms, and the humans are eukaryotic organisms and this is the reason of essential difficulties in developing the selectively acting drugs with low toxicity for the patients. This difficulty has been omitted in the treatment of topical fungal infections. This includes such areas of clinical mycology as gynecology, dermatology, gastroenterology, pulmomology, urology and ophthalmology, where the problem of compounds toxicity occurs to be less dramatic (C. P. Schaffner, in Macrolide antibiotics, S. Omura (ed.), Academic Press. Inc., Orlando, p. 457, 1984). The most popular drugs from polyene group for such treatment are Nystatin, Pimaricin and Amphotericin B, which due to the lack of resorption in oral administration are practically non-toxic. However, invasive mycoses concerning the infection of internal organs and fungemia are still problems far from successful solution. Current epidemiological statistics concerning the mortality in such types of illnesses are not satisfactory (M. A. Pfaller, D. J. Diekem, Clin. Microbiol. Rev. 20, 133, 2007; T. F. Patterson, Lancet 366, 1013, 2005; S. K Fridkin, Clin. Infect. Dis. 41, 240, 2005). Especially dangerous are invasive candidoses and aspergilloses and infections caused by certain other fungal pathogenes. In the case of invasive candidosis the mortality is in the range 30-70%, aspergillosis more than 50%, the frequency of invasive mycoses in oncology/hematology is approximately 50%, in the case of mycoses of children with leukemia is 29-39% (S. E. Soloviera et al., Rus. Chem. Rev. 80, 103, 2011: A. L. Demain, S. Sanchez, J. Antibiot., 62, 5, 2009; G. O. Bronin et al., Pediatryia 4, 31, 2004). Over 90% of HIV-positive patients suffer from mycoses, while pneumonia caused by Pneumocystis carinii is the main reason of death in patients with AIDS. Systemic mycoses are common reason of death in adult patients with leukemia. Candida spp., in regard to frequency of incidences, are the fourth etiological factor of hospital infections and is a cause of 8-11% of all general infection with mortality up to 40%. The frequency of fungal infections in patients after organ transplants is in the range 5-40% depending on the type of organ transplanted. Invasive aspergilosis of lungs is main reason of patients death after transplant of bone marrow. Blastomycosis, histoplasmosis and coccidiomycosis are endemic mycoses with high frequency of appearance in many regions of the world.
The unfavourable situation in the clinical mycology since over 20 years is constantly getting worse for several reasons. One of them is steady increase of infections caused by species of fungal microorganisms previously being non-pathogenic (D. A. Enoch et al., J. Med. Microbiol. 55, 809, 2006; N. Nucii, K. A. Man, Clin. Infect Dis. 41, 521, 2005). The increase of fungal infections is also caused by the use of antibacterial chemotherapeutical agents with broad spectrum and by the use of steroids, and above all by decreasing the immune system activity in an increasing number of patients, connected with the development of transplantology, which required the use of immunosuppressive drugs, and also with the increase of cancer cases and thus usage of immunosuppressive cytostatics (N. Siugh, Med. Mycol. 43, suppl. 1, 267, 2005; A. L. Demain, S. Sanchez, J. Antibiot. 62, 5, 2009).
Especially alarming is the steady decrease of therapeutic values of currently available antifungal chemotherapeutics used for the treatment of systemic infections, as a result of the rapid development of resistance of pathogenic fungal strains (D. Sanglard, Curr. Opinion Microbiol. 5, 378, 2002; D. P. Kontoyiannis, R. E. Lewis, Lancet 359, 1135, 2002). The most dangerous type of fungal resistance is the multidrug resistance (MDR), which affects antifunagals activity towards systemic as well as topical administration. The MDR strains overexpress transporter proteins which export a drug from the cells, thus not allowing to retain in fungal cells its therapeutic concentration.
Enzyme inhibitor, 5-fluorocytosine, often used in combination with Amphotericin B to increase its uptake by membrane permeabilisation, as an antimetabolite avoids the exporting activity of MDR transporting proteins, but enhances the development of specific type of resistance mainly by the loss of cytosine permease and deaminase. Particularly clinically valuable fungicides of “azoles” group, mostly triazoles, such as flucanazole, voriconazole, posaconazole and others, are partially susceptible to their removal from the cells by MDR exporting proteins (R. Franz et al., Antimicrob. Ag. Chemother., 42, 3065, 1998; R. Wakiec et al., Mycoses, 50, 109, 2007). However, being the inhibitors of lanosterol demethylase, interacting with the enzyme, also induce the changes in the structure of enzymatic protein leading to the loss of inhibitory activity of these compounds. Valuable and very promising fungicide caspofungin, although with narrow antifungal spectrum but with excellent selectivity, as inhibitor of β-D-glucan synthase, interacts with the enzyme, which unfortunately leads to the induction of changes in the structure of enzyme protein and in consequence the loss of inhibitory activity of compound. The reports on growing resistance to the action of this drug are starting to be published. Therefore, concerning the MDR problem, there practically remains only one effective group of fungicides, polyene macrolides, which do not induce the development of resistant strains. These antibiotics as not being the substrates of MDR exporting proteins, retain full activity against multidrug resistant strains (M. Ślisz i in., J Antibiot. 60, 436, 2007). Although there is data on the appearance of strains with reduced sensitivity to these antibiotics, as a result of certain changes in the lipid composition of cytoplasmic membrane, but these changes are rather phenotypic and regressing after discontinued contact with the drug.
Presented situation in clinical mycology points to the necessity of further search for antifungal drugs. One of the intensively developing research projects concerned study on the modifications of polyene macrolides aimed at removing of their main shortcomings, which are high toxicity and lack of water solubility of native compounds. However, until now none of the products of this antibiotics modification have been introduced to clinical practice. The only practical progress in this area was the introduction to clinical use of Amphotericin B complexes with lipids or liposomal formulations as Abalcet®, Amphotec® and AmBisome®. However, these formulations of Amphotericin B are only a little less toxic in comparison to the native antibiotic.
Due to the high toxicity of Amphotericin B, as well as its very high cost, the attention has been drawn by another clinical antibiotic from polyenes group, Nystatin, so far used only topically. This antibiotic exhibits all advantageous features of that type of compounds such as: broad antifungal spectrum, fungicidal effect, high antifungal activity, lack of induction of resistance and lack of interaction with overexpressed protein transporters in multidrug resistant strains (MDR). Furthermore, Nystatin is much cheaper than Amphotericin B. Studies have been undertaken on the application of Nystatin also in the treatment of systemic fungal infections, using soluble lipidic and lyposomal formulations (R. Semis et al., Mycopathologia, 169, 333, 2010; R. Semis et al., J. Antimicr. Ag., 38, 336, 2011; R. Semis et al., J. Antimicrob. Chemother., 67, 1716, 2012; R. Semis et al., Med. Mycol. Month Early on line 1, 2012).
The studies on chemical modifications of Nystatin have been also carried out. Earlier obtained Nystatin A1 derivatives, are compounds with the modified amino group of the mycosamine moiety and also the carboxyl group of aglycone. The attempts to modify compounds by genetic manipulations of antibiotic-producing organism have also been performed. Chemical modifications of Nystatin A1 have been intended to improve the solubility and to reduce toxicity of the compounds. However, no significant progress has been achieved in this matter, because no theoretical background for the rational modifications has been worked out. The synthesis of derivatives had accidental character and were based rather on random screening.
The prior art on Nystatin A1 derivatives includes 1) derivatives at amino group; 2) derivatives at carboxyl group; 3) double derivatives including amino and carboxyl group; 4) analogues with genetically modified aglycone fragment.
Among the derivatives at amino group many N-acyl derivatives have been synthesized. There are simple derivatives (U.S. Pat. No. 3,244,590; C. P. Schaffner, E. Borowski, Antibiot. Chemother. 11, 724, 1961), more complicated ones (L. Silva et al., J. Photochem. Photobiol. B. Biol. 72, 17, 2003), compounds including fluoroorganic derivatives (Yu. Shenin et al., Pharmac. J. 32, 109, 1996), amino acyl derivatives (A. Czerwiński., J. Antibiot. 39, 1025, 1986; Polish Patent 142847). Other derivatives at amino group comprise guanidine derivative (U.S. Pat. No. 4,396,610), N-enamine and amidine derivatives (Stefańska et al., Acta Polon. Pharm., XLV, 71, 1988; Polish Patent 120111), N-trialkylsilyl derivatives (V. V. Balakhov et al., Khim. Farm. Zh. 11, 45, 1977) and Schiff bases (V. V. Balakhov et al., Khim. Farm. Zh. 11, 45, 1977). A large group constitutes N-alkyl derivatives. These are N-alkylhydrofosforyl derivatives (V. V. Belakhov et al., Khim. Farm. Zh. 25, 45, 1991), N,N,N-trimethyl derivative (U.S. Pat. No. 4,144,328), products of reductive N-alkylation (V. Paquet, E. M. Carreira, Org. Lett. 8, 1807, 2006; U.S. Pat. No. 6,664,241 B2). Particular attention is drawn to N-alkyl derivatives which are N-glycosyl products of Amadori reaction rearrangement (L. Falkowski et al., J. Antibiot. 28, 244, 1975; L. Falkowski et al., Acta Polon. Pharm. 37, 517, 1980; L. Falkowski et al., Polish J. Chem. 56, 123, 1982; Polish Patent 82224; U.S. Pat. No. 6,664,241 B2) and their water-soluble salts with N-methyl-D-glucamine (U.S. Pat. No. 4,195,172).
Nystatin A1 derivatives with modified carboxyl group include a number of compounds. These are esters (T. Bruzzesse et al., Experientia 28, 1515, 1972; T. Bruzzesse et al., J. Pharm. Sci. 64, 462, 1975; U.S. Pat. No. 3,780,173; P. Schaffner, W. Mechlinski, J. Antibiot. 25, 259, 1972; D. P. Bonner et al., J. Antibiot. 25, 261, 1972; B. Stefańska et al., Acta Polon. Pharm. 40, 71, 1983; U.S. Pat. No. 5,981,721), hydrazides (J. Grzybowska, E. Borowski J. Antibiot. 43, 907, 1990) and amides (Polish Patent 138831; U.S. Pat. No. 6,664,241 B2), as well their soluble salts (Polish Patent 138831).
There are also known Nystatin A1 derivatives modified in both amino and carboxyl groups of the antibiotic. These compounds combine above described types of chemical modification at both groups. They include the methyl ester of N,N,N-trimethyl derivative (L. Falkowski et al., J. Antibiot. 32, 1080, 1979; L. Falkowski et al., Acta Polon. Pharm., 37, 631, 1980; Polish Patent 122884), methyl esters of N-enamine and amidine derivatives (Polish Patent 120035), esters of guanidine derivatives (U.S. Pat. No. 4,396,610), methyl ester of N-fructosyl-N-methyl Nystatin A1 (Polish patent 199213), esters of glycosyl derivatives (U.S. Pat. No. 6,562,796 B2), methyl esters of aminoacyl derivatives (Polish Patent 142848) and amides of N-mono and di-alkyl, as well of N-glycosyl derivatives.
Efforts have been also made to modify the macrolide ring of Nystatin A1 by the genetic manipulation of Streptomyces noursei, which produces this antibiotic. That way two new hydroxyl groups have been introduced to that ring, which slighty increase the hydrophilicity of the molecule and thus improve its solubility in water (S. F. E. Borgos et al., J. Med. Chem. 49, 2431, 2006). However, these derivatives cannot be regarded as derivatives of Nystatin A1, but as its analogs. Similarly, other genetic modifications of the antibiotic “producer” also lead to obtaining novel antibiotics and their derivatives, which cannot be classified as a semi-synthetic derivatives of the original native product, Nystatin A1 (International Patent Application PCT/GB2008/002238). Other known Nystatin derivatives are presented in the patent specification WO 01/68102, and in U.S. Pat. No. 6,413,537.
Among above mentioned Nystatin A1 derivatives, there are no compounds which could combine features, particularly important for practical application, such as water solubility and essentially reduced toxicity.
The advantage of new semisynthetic Nystatin A1 derivatives, according to the invention, is that they exhibit low hemotoxicity, which is a common toxicity test for compounds of polyene macrolides group, and form water soluble salts. They also exhibit antifungal activity towards broad spectrum of Candida species, filamentous fungi and dermatophytes and against strains with multidrug resistance (MDR) with overexpression of MDR protein transporters MDR1p, as well as Cdr1p and Cdr2p.
Unexpected novelty enabling to obtain according to the invention, advantageous effect of hemotoxicity reduction and water solubility, is introduction to substituents at amino group of Nystatin A1 of bulky moieties, which induce steric hindrance effects. It appeared that such steric hindrance factors decrease lethal permeabilising activity of Nystatin A1 derivatives a greater degree towards mammalian than fungal cells, which increases their selectively of action and essentially reduces the hemotoxicity of these compounds. The presence of a bulky moiety attached to the amino group of Nystatin A1 also disturbs the zwitterionic structure of the antibiotic thus enabling the formation of soluble salts.
Bulky moieties which may give the effect of steric hindrance include ring systems carbo-, as well as heterocyclic, alicyclic and aromatic, bulky substituents as tert-butyl, nitro group, bromine atom and also aliphatic fragments, which, due to their flexibility, can form voluminous conformational structures. The introduction of the steric hindering moieties to the molecule of a biologically active compounds can influence in a different way the affinity to its molecular target, which in the case of Nystatin A1 and its derivatives is ergosterol in fungal cells and cholesterol in mammalian cells. Until now, for Nystatin A1 derivatives, the influence of steric hindrance on the differentiation of lethal effects towards both types of cells has not been known. There is no general rule concerning the influence of steric hindrance of biologically active compounds on their properties. This effect is associated with a defined compound with a specific structure and its conformational dynamics. Nystatin A1 molecule, because of its flexible structure is very dynamic in regard to conformational changes. Effect of steric hindrance of derivatives on their biological properties can be confronted only with other polyene macrolides of the same group which are tetraeno-dienes. In that respect, there is no data available prior to the invention.